Melanoma is a highly aggressive skin tumor that originates in the epidermis from melanocytes. As melanocytes share with the nervous system a common neuroectodermal origin and express all neurotrophins (NTs), we evaluated the expression and function of NTs and their receptors in melanoma. We report that primary and metastatic melanoma cell lines synthesize and secrete all NTs. Moreover, melanoma cells express the low-affinity (p75NTR) and the high-affinity tyrosine kinase NT receptors (Trk). The inhibition of Trk receptors by either K252a or Trk/Fc chimeras prevents proliferation, indicating that autocrine NTs are responsible for this effect. NT-3, NT-4, and nerve growth factor (NGF) induce cell migration, with a stronger effect on metastatic cell lines. Transfection with p75NTR small interfering RNA (p75NTRsiRNA) or treatment with K252a inhibits NT-induced melanoma cell migration, indicating that both the low- and high-affinity NT receptors mediate this effect. All melanoma cell lines express the p75NTR coreceptor sortilin by which proNGF stimulates migration in melanoma cells, but not in cells transfected with p75NTRsiRNA. These results indicate that NTs, through their receptors, play a critical role in the progression of melanoma.

Survivin belongs to the family of inhibitor of apoptosis proteins and is involved in regulation of cell death as well as cell division. Here, we show that wild-type (WT) survivin is expressed in a subpopulation of basal keratinocytes in normal human skin at the cytoplasmic level. WT survivin is highly expressed in keratinocyte stem cells (KSCs), whereas its mRNA level decreases in transit amplifying (TA) cells and disappears in postmitotic (PM) cells. Likewise, WT survivin protein is expressed in KSCs, almost undetectable in TA cells, and absent in PM cells. Real time polymerase chain reaction demonstrates that the putative antiapoptotic isoforms survivin-2B and survivin-⌬Ex3 are expressed at the highest levels in KSCs, whereas they tend to decrease in TA cells and disappear in PM cells. On the contrary, the putative proapoptotic variants of survivin, survivin-3B, and survivin-2␣ tend to be high in PM and TA cells and are almost absent in KSCs. By confocal microscopy, survivin is predominantly expressed at the nuclear level in KSCs, which proliferate significantly better than TA cells, which, in turn, express mostly cytosolic WT survivin. Blocking ␤1 integrin signal downregulates WT survivin mRNA and protein expression and induces apoptosis (anoikis) in KSCs. On the other hand, inhibition of ␤1 integrin upregulates mRNA expression of survivin-2␣. Taken together, these results indicate that survivin identifies human KSCs. Expression of nuclear survivin could reflect the different behavior between KSCs in vitro and in vivo, in terms of proliferation. Finally, survivin could be part of the "niche" protection by preventing anoikis in KSCs. STEM CELLS 2007;25:149 -155

Phosphodiesterases 4 (PDE4) act as proinflammatory enzymes via degradation of cAMP, whereas PDE4 inhibitors play an anti-inflammatory role in vitro and in vivo. In particular, apremilast has been recently approved for the treatment of psoriasis and psoriatic arthritis. However, little is known on the expression pattern of PDE4 in psoriasis. We report that PDE4B and PDE4D mRNA are overexpressed in peripheral blood mononuclear cells (PBMC) from psoriasis, as compared with normal controls, while apremilast reduces PBMC production of a number of pro-inflammatory cytokines and increases the levels of anti-inflammatory mediators. PDE4 expression is up-regulated in psoriatic dermis as compared with normal skin, with particular regard to fibroblasts. This is confirmed in vitro, where both dermal fibroblasts (DF) and, to a greater extent, myofibroblasts (DM) express all PDE4 isoforms at the mRNA and protein level. Because PDE4 interacts with the nerve growth factor (NGF) receptor CD271 in lung fibroblasts, we evaluated the relationship and function of PDE4 and CD271 in normal human skin fibroblasts. All PDE4 isoforms co-immunoprecipitate with CD271 in DM, while apremilast inhibits apoptosis induced by β-amyloid, a CD271 ligand, in DM. Furthermore, apremilast significantly reduces NGF- and transforming growth factor-β1 (TGF-β1)-induced fibroblast migration, and inhibits DF differentiation into DM mediated by NGF or TGF-β1. Finally, in DM, apremilast significantly reduces cAMP degradation induced by treatment with β-amyloid. Taken together, these results indicate that PDE4 play an important role in psoriasis. In addition, the study reveals that the PDE4/CD271 complex could be important in modulating fibroblast functions.

β1-integrin protects keratinocyte stem cells (KSC) from cell-detachment apoptosis (`anoikis'). Here we show that caspase-8 active protein is detected in both young transit amplifying (TA) cells and TA cells, but not in KSC. On suspension, caspases are activated earlier in young TA than in KSC, whereas anti-β1-integrin neutralizing antibody accelerates caspase activation in both KSC and young TA. Caspases 8 and 10 are the first caspases to be activated whereas caspase-8 inhibitor zIETD-fmk delays the activation of Bid, caspase-9 and caspase-3. However, the caspase-9 inhibitor zLEDH-fmk does not block the activation of caspase-8, Bid, caspase-10 and caspase-3. Moreover, caspase-8, but not caspase-9 inhibitor partially prevents keratinocyte anoikis. As FLIP inhibits caspase-8 processing, we retrovirally infected HaCaT keratinocytes with c-FLIPL. Anti-β1-integrin fails to activate caspase-8, Bid, caspase-9 and to induce the release of cytochrome c in c-FLIPL overexpressing keratinocytes. Finally, overexpression of c-FLIPL partially prevents anoikis in both suspended and anti-β1 integrin-treated cells. Taken together, these results indicate that the extrinsic apoptotic pathway triggered by caspase-8 predominates in keratinocyte anoikis. However, the release of cytochrome c and the later activation of caspase-9 seem to suggest that the intrinsic mitochondrial pathway may intervene as a positive feedback loop of caspase activation.

p75 neurotrophin receptor (p75NTR) belongs to the TNF-receptor superfamily and signals apoptosis in many cell settings. In human epidermis, p75NTR is mostly confined to the transit-amplifying (TA) sub-population of basal keratinocytes. Brainderived neurotrophic factor (BDNF) or neurotrophin-4 (NT-4), which signals through p75NTR, induces keratinocyte apoptosis, whereas b-amyloid, a ligand for p75NTR, triggers caspase-3 activation to a greater extent in p75NTR transfected cells. Moreover, p75NTR co-immunoprecipitates with NRAGE, induces the phosphorylation of c-Jun N-terminal kinase (JNK) and reduces nuclear factor kappa B (NF-jB) DNA-binding activity. p75NTR also mediates pro-NGF-induced keratinocyte apoptosis through its co-receptor sortilin. Furthermore, BDNF or b-amyloid cause cell death in TA, but not in keratinocyte stem cells (KSCs) or in p75NTR silenced TA cells. p75NTR is absent in lesional psoriatic skin and p75NTR levels are significantly lower in psoriatic than in normal TA keratinocytes. The rate of apoptosis in psoriatic TA cells is significantly lower than in normal TA cells. BDNF or b-amyloid fail to induce apoptosis in psoriatic TA cells, and p75NTR retroviral infection restores BDNF-or b-amyloid-induced apoptosis in psoriatic keratinocytes. These results demonstrate that p75NTR has a pro-apoptotic role in keratinocytes and is involved in the maintenance of epidermal homeostasis.

Neurotrophins (NTs) belong to a family of growth factors that play a critical role in the control of skin homeostasis. NTs act through the low-affinity receptor p75NTR and the high-affinity receptors TrkA, TrkB, and TrkC. Here we show that dermal fibroblasts (DF) and myofibroblasts (DM) synthesize and secrete all NTs and express NT receptors. NTs induce differentiation of DF into DM, as shown by the expression of α-SMA protein. The Trk inhibitor K252a, TrkA/Fc, TrkB/Fc, or TrkC/Fc chimera prevents DF and DM proliferation. In addition, p75NTR siRNA inhibits DF proliferation, indicating that both NT receptors mediate DF proliferation induced by endogenous NTs. Autocrine NTs also induce DF migration through p75NTR and Trk, as either silencing of p75NTR or Trk/Fc chimeras prevent this effect, in absence of exogenous NTs. Finally, NGF or BDNF statistically increase the tensile strength in a dose dependent manner, as measured in a collagen gel through the GlaSbox device. Taken together, these results indicate that NTs exert a critical role on fibroblast and could be involved in tissue re-modeling and wound healing.

Epidermal fatty acid-binding protein (E-FABP) is a lipid carrier, originally discovered in human epidermis. We show that E-FABP is almost exclusively expressed in postmitotic (PM) keratinocytes, corresponding to its localization in the highest suprabasal layers, while it is barely expressed in keratinocyte stem cells (KSC) and transit amplifying (TA) keratinocytes. Transfection of normal human keratinocytes with recombinant (r) E-FABP induces overexpression of K10 and involucrin. On the other hand, E-FABP inhibition by siRNA downregulates K10 and involucrin expression in normal keratinocytes through NF-κB and JNK signalling pathways. E-FABP is highly expressed in psoriatic epidermis, and it is mainly localized in stratum spinosum. Psoriatic PM keratinocytes overexpress E-FABP as compared to the same population in normal epidermis. E-FABP inhibition in psoriatic keratinocytes markedly reduces differentiation, while it upregulates psoriatic markers such as survivin and K16. However, under high-calcium conditions, E-FABP silencing downregulates K10 and involucrin, while survivin and K16 expression is completely abolished. These data strongly indicate that E-FABP plays an important role in keratinocyte differentiation. Moreover, E-FABP modulates differentiation in psoriatic keratinocytes.

CD271 is the low-affinity neurotrophin (p75NTR) receptor that belongs to the tumor necrosis factor receptor superfamily. Because in human epidermis, CD271 is predominantly expressed in transit-amplifying (TA) cells, we evaluated the role of this receptor in keratinocyte differentiation and in the transition from keratinocyte stem cells (KSCs) to progeny. Calcium induced an upregulation of CD271 in subconfluent keratinocytes, which was prevented by CD271 small interfering RNA. Furthermore, CD271 overexpression provoked the switch of KSCs to TA cells, whereas silencing CD271 induced TA cells to revert to a KSC phenotype, as shown by the expression of β1-integrin and by the increased clonogenic ability. CD271(+) keratinocytes sorted from freshly isolated TA cells expressed more survivin and keratin 15 (K15) compared with CD271(-) cells and displayed a higher proliferative capacity. Early differentiation markers and K15 were more expressed in the skin equivalent generated from CD271(+) TA than from those derived from CD271(-) TA cells. By contrast, late differentiation markers were more expressed in skin equivalents from CD271(-) than in reconstructs from CD271(+) TA cells. Finally, skin equivalents originated from CD271(-) TA cells displayed a psoriatic phenotype. These results indicate that CD271 is critical for keratinocyte differentiation and regulates the transition from KSCs to TA cells.